Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
This invention provides an improved method for purification of recombinant copper/zinc (Cu-Zn) superoxide dismutase from bacteria or eucaryotic cells.
The clinical potential of the enzyme superoxide dismutase (SOD) is enormous. The scientific literature suggests that SOD could be useful in a wide range of clinical applications. These include prevention of oncogenesis and tumor growth and reduction of cytotoxic and cardiotoxic effects of anticancer drugs, anti-inflammatory protection of ischemic tissues and protection of spermatozoa. In addition, there is great interest in studying the effect of SOD on the aging process. There is also evidence that SOD can be used to prevent the damage sometimes caused to newborn babies by hyperbaric oxygen treatment.
In particular, there is increasing evidence that "reperfusion injury" caused by oxygen free radicals (probably generated from xanthine oxidase) accounts for much of the damage caused by a period of ischemia in many organs of the body such as the spinal cord, intestine, skin, heart, lung, pancreas or kidney (11).
In vitro and animal experiments using human recombinant copper/zinc superoxide dismutase (hCu-Zn SOD analog), produced as described in coassigned U.S. Pat. No. 4,742,004, issued May 2, 1988, and in corresponding European patent application publication no. 0173280, published May 5, 1986, have demonstrated the efficacy of SOD in reducing reperfusion injury e.g., in the spinal cord of dogs (12), in the hearts of rabbits (13), and in the hearts of dogs (14).
The superoxide anion (0.degree..sub.2) produced on reperfusion of ischemic tissue and also in certain inflammatory conditions is highly toxic to macromolecules, e.g., 0.degree..sub.2 may react with lipid hydroperoxides to form alkoxy radicals in phospholipid membranes.
The superoxide anion (0.degree..sub.2) is removed in normal biologic tissue by the dismutation reaction: EQU 20.degree..sub.2 +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2
The above reaction can proceed spontaneously or it can be catalyzed by SOD which increases the rate of intercellular dismutation by a factor of 10.sup.9. SOD is a ubiquitous mammalian enzyme. In the presence of normal intercellular concentrations of catalase and peroxidase, SOD is responsible for the "scavenging" of oxygen-free radicals, thereby serving as a normal biological defense against the formation and accumulation of reduced oxygen intermediates.
Copper/zinc (Cu-Zn) SOD has been demonstrated in virtually all eucaryotic organisms. Human Cu-Zn SOD-1 is a dimeric metallo-protein composed of identical non-covalently linked subunits, each having a molecular weight of 16,000 daltons and containing one atom of copper and one of zinc. Each subunit is composed of 153 amino acids whose sequence has been established.
Endogenous SOD is present in tissues in limited amounts and when high levels of superoxide anion are produced, the amount of SOD present is not sufficient. Thus there is a need for clinical administration of exogenous SOD. However, exploration of the therapeutic potential of human SOD-1 (EC 1.15.1.1) has been limited mainly due to its scarce availability. To overcome this problem we inserted an SOD clone containing the entire coding region of human SOD-1 described by Groner and his colleagues (15) into efficient bacterial expression vectors which express hSOD analog at high levels; this has been described in coassigned U.S. Pat. No. 4,742,004 described above. Using this method, we have produced recombinant hSOD analog which differs from authentic human SOD (from blood) in that the amino terminus alanine is not acetylated. The amino acid sequence of the bacterial-produced SOD analog does not contain a methionine residue at its N-terminus.
The recombinant hSOD analog thus produced is very pure. However, it was desirable to produce even higher levels of purity. An improved chromatography method which produces an even higher degree of purification, surprisingly achieved without loss of yield, is described in this application. A novel "exchange" procedure which increases the yield of pure recombinant SOD produced, without loss of purity, is also described.
European patent application publication no. 0180964, published May 14, 1986 and assigned to Ube Industries Limited, discloses the production of human Cu-Zn SOD in Escherichia coli and partial purification of the hSOD produced. This method was done on small scale only (20 g of wet cells). No details of purification achieved are given apart from specific activity. There is no disclosure of the extent of contamination by Escherichia coli proteins and endotoxins.
European patent application publication no. 0138111, published Apr. 24, 1985 and assigned to Chiron Corporation, discloses the production of hSOD in both Escherichia coli and yeast, but no methods of production of hSOD from crude cell lysates are disclosed. European patent application publication no. 0164556, also assigned to Chiron, discloses the production of a yeast expression plasmid for hSOD, but again no methods of purification of hSOD are disclosed.
Hallewell et al. from Chiron Corporation (16) disclose production of recombinant SOD but merely state that the recombinant SOD was purified to homogeneity by conventional means after lysing the yeast cells with glass beads and pelleting the cell debris.
Hallewell et al. from Chiron Corporation (17) disclose a method of purifying recombinant SOD from yeast cells. This method gives no detail of the yield of hSOD produced, nor its degree of purification; it is also a purification method of hSOD analog from yeast cells and not Escherichia coli cells.
Takahara et al. (18) disclose the secretion by Escherichia coli cells of hSOD into the periplasmic space. This is a very small-scale procedure (16 ml medium), and the SOD produced is not purified; the Escherichia coli cells are simply subjected to osmotic shock, and subcellular fractions are analyzed by SDS-PAGE.